PROJECTSUMMARY TheProteomicsandMetabolomicsFacility,supportedbytheCancerCenterSupportGrant(CCSG)awarded toTheWistarInstitute,providesacomprehensivesetofproteomicsandtargetedquantitativemetabolite assaystotheWistarCancerCentermembershipasaprimarygoal.ResourcesofthisFacilityarealso availabletoinvestigatorsinotherCancerCentersandotheracademicinvestigatorsasasecondarygoal.Dr. Tang?sroleastheFacilityManagingDirectoristosupportcancerresearchandotherbiomedicalresearchby providingexpertconsultationandstate-of-the-arttechnologiesthatoperateatmaximumperformanceat affordablecoststousers.TheManagingDirectorwillassistinexperimentaldesign,manageFacilitystaff, performMSdataanalysesasneeded,andassistinthebiologicalinterpretationofresults.TheManaging Directorwillalsodevotesubstantialefforttooptimizingandimplementingnewmethods,updateanalyticaland dataanalysesmethods,andupdateinstrumentationtoensureeachprojectisperformedusingstate-of-the-art methodologies.Thisiscriticalbecauseinstrumentation,software,andanalyticalstrategiescontinuetoevolve rapidly,andmostcurrentandanticipatedfutureprojectsinvolveverychallengingproteomicsand metabolomicsproblems.Proteomicsprojectswillinclude:1)LC-MS/MSanalysisofisolatedproteincomplexes withandwithoutchemicalcrosslinking,2)in-depth,global,quantitativecomparisonsofexosomes,secretomes, andcelllysates,and3)targetedandglobalquantitativecomparisonsofposttranslationalmodifications. Quantitativedatawillbeobtainedeitherusinglabel-freequantitationofintegratedMSioncurrents,or quantitationusingstableisotopessuchasSILACorisobarictag(TMToriTRAQ)labeling.Theproteome analyseswilluseoptimizedmethodsandstate-of-the-artinstruments,suchastheThermoScientificQ ExactiveHFmassspectrometer,thatcandetectandrobustlyquantifymostoftheproteinspresentincomplex samples,includingwholecelllysates.Metabolomicsprojectswillincludequantifyingthesteadystatelevelsof keymetabolitesusingtargetedmultiplexMRM-MSontheSciexQTRAP5500hybridtriplequadrupolelinear iontrapmassspectrometer.Plansforfuturedevelopmentinclude:1)expandingdepthofproteomeanalyses toefficientlyanalyzegreaterthan90%ofgeneproductsexpressedbycellsandtissues,2)implementing routineuseofchemicalcross-linkingtoidentifyprotein-proteininteractionsandobtainstructuralinformation,3) implementationofmetabolitefluxanalysesusing13Cstableisotopetracer,and4)implementationoflipidomics analyses.AllofthesenewmethodswillusetheThermoScientificQExactivemassspectrometersorfuture nextgenerationinstruments.Theseproteomicsandmetabolomicsanalyseswillcontributetocriticaldata requiredtoidentifyproteinandmetabolitetargets,aswellasgeneratehypothesesthatarevitalforthesuccess ofthecancer-relatedprojectsdescribedinthisapplication.